Methods to impair hematologic cancer progenitor cells and compounds related thereto

ABSTRACT

Primitive or progenitor hematologic cancer cells have been implicated in the early stages and development of leukemia and malignant lymphoproliferative disorders, including acute myelogenous leukemia (AML), chronic myelogenous leukemia (CML) and chronic lymphoid leukemia (CLL). Interleukin-3 receptor alpha chain (IL-3Rα or CD123) is strongly expressed on progenitor hematologic cancer cells, but is virtually undetectable on normal bone marrow cells. The present invention provides methods of impairing progenitor hematologic cancer (e.g., leukemia and lymphomic) cells by selectively targeting cells expressing CD123. These methods are useful in the detection and treatment of leukemias and malignant lymphoproliferative disorders. Also provided are compounds useful for selectively binding to CD123 and impairing progenitor hematologic cancer cells. These compounds may include cytotoxic moieties such as, for example, radioisotopes or chemotherapeutics.

CONTINUING DATA

The present application is a continuation of U.S. application Ser. No.10/830,089, filed Apr. 23, 2004, now U.S. Pat. No. 7,651,678, which is acontinuation of U.S. application Ser. No. 09/799,100, filed Mar. 6,2001, now U.S. Pat. No. 6,733,743, which claims the benefit of priorityto U.S. Provisional Patent Application Nos. 60/187,123, filed Mar. 6,2000, and 60/227,295, filed Aug. 24, 2000, the disclosures of each ofwhich are incorporated by reference herein in their entirety.

BACKGROUND OF THE INVENTION

1. Field of the Invention

The present invention is related to methods of impairing progenitorhematologic cancer cells or treating hematologic cancer by targeting acell surface marker specific for progenitor hematologic cancer cells.The present invention is also related to a method for diagnosinghematologic cancer.

2. Background of the Invention

Stem cells are commonly found in a variety of mammalian tissue systems.While the criteria by which such cells are defined vary depending uponthe specific context, two properties are generally regarded as centralfeatures of stem cell populations: (1) stem cells must exhibit somecapacity for self-replication or self-renewal, and (2) stem cells mustbe capable of differentiating into appropriate lineages (Potten C S:Stem Cells. London, Academic Press, 1997). Cells of this nature havebeen described for a number of tissues including hematopoietic,embryonic, neural, muscle and hepatic systems (Lemischka I R. Clonal, invivo behavior of the totipotent hematopoietic stem cell. Semin Immunol1991, 3: 349-55; Morrison S J, et al., The biology of hematopoietic stemcells. Annu. Rev. Cell Dev. Biol. 1995, 11: 35-71; Robertson E J., Usingembryonic stem cells to introduce mutations into the mouse germ line.Biol Reprod 1991, 44: 238-45; Gage F H., Mammalian neural stem cells.Science 2000, 287: 1433-8; and, Alison M, et al., Hepatic stem cells. JHepatal 1998, 29: 676-82). Thus, it is perhaps not surprising thatsimilar cells have recently been documented in the context of malignantpopulations (Bonnet D, et al., Human acute myeloid leukemia is organizedas a hierarchy that originates from a primitive hematopoietic cell. Nat.Med. 1997, 3: 730-737; Blair A, et al., Most acute myeloid leukemiaprogenitor cells with long-term proliferative ability in vitro and invivo have the phenotype CD34(+)/CD71(−)/HLA-DR−. Blood 1998, 92:4325-35; Cobaleda C, et al., A primitive hematopoietic cell is thetarget for the leukemic transformation in human Philadelphia-positiveacute lymphoblastic leukemia. Blood 2000, 95: 1007-13). Indeed, a stemcell is in some respects the ideal target for malignant transformationin that relatively little biological change is required. Since stemcells already possess the genetic programming necessary to be highlyproliferative and developmentally plastic, one can imagine thatrelatively subtle perturbations might be sufficient to induce disease.

One example of neoplasia arising from malignant stem cells has recentlybeen documented in the hematopoietic system in the case of acutemyelogenous leukemia (AML). This disease is characterized by prematurearrest of myeloid development and the subsequent accumulation of largenumbers of non-functional leukemic blasts. While leukemic blast cellsare often of clonal origin and display relatively homogeneous features,it has been demonstrated that such populations are organized in ahierarchical fashion, analogous to normal hematopoietic progenitors.Thus, there is a phenotypically defined leukemic stem cell populationthat is sufficient to propagate leukemic blasts both in vitro and invivo in xenogeneic mouse models of human AML (Bonnet D, et al., Humanacute myeloid leukemia is organized as a hierarchy that originates froma primitive hematopoietic cell. Nat. Med. 1997, 3: 730-737; Blair A, etal., Most acute myeloid leukemia progenitor cells with long-termproliferative ability in vitro and in vivo have the phenotypeCD34(+)/CD71(−)/HLA-DR−. Blood 1998, 92: 4325-35, Cobaleda C, et al., Aprimitive hematopoietic cell is the target for the leukemictransformation in human Philadelphia-positive acute lymphoblasticleukemia. Blood 2000, 95: 1007-13; Blair A, et al. Lack of expression ofThy-1 (CD90) on acute myeloid leukemia cells with long-termproliferative ability in vitro and in vivo. Blood 1997, 89: 3104-12).The concept of a leukemic stem cell (LSC) becomes critically importantin considering the etiology of human disease. Clearly, in order toachieve durable remission, it will be necessary to specifically ablatethe primitive or progenitor LSC population. However, previous studies(Terpstra W, et al., Fluorouracil selectively spares acute myeloidleukemia cells with long-term growth abilities in immunodeficient miceand in culture. Blood 1996, 88: 1944-50), as well as data from ourgroup, suggest that LSC's are biologically distinct from more matureleukemic blasts and may not be responsive to conventionalchemotherapeutic regimens. This observation is consistent with theclinical profile frequently seen for AML, wherein a majority of patientscan achieve apparent complete remission, but in most cases will relapse(Schiller G J., Treatment of resistant disease. Leukemia 1998, 12 Suppl1: S20-4; Paietta E., Classical multidrug resistance in acute myeloidleukemia. Med Oncol 1997, 14: 53-60). If LSC's are more refractile tochemotherapy than blasts, it is attractive to propose that survivingstem cells are a major contributing factor to leukemic relapse. Thus,strategies that specifically target progenitor leukemia cells mayprovide more effective treatment for leukemia patients. In 1997, Bonnetand Dick described the phenotype for LSC's as CD34+/CD38− (Bonnet D, etal., Human acute myeloid leukemia is organized as a hierarchy thatoriginates from a primitive hematopoietic cell. Nat. Med. 1997, 3:730-737). We report that the IL-3 receptor alpha chain (CD123) is highlyexpressed on leukemic but not normal CD34+/CD38− hematopoietic cells. Inview of this state of the art, there is a need in the art to provide adiagnostic method for detecting leukemia at an early stage, as well asmore effective methods of treating this disease.

SUMMARY OF THE INVENTION

The present invention relates to a method of using compounds that bindto the human CD123 molecule (CD123 ectopeptide), in the diagnosis andtreatment of hematologic cancers (e.g., leukemias and malignantlymphoproliferative disorders). The CD123 specific compounds andmimetics have particular utility as pharmaceuticals and reagents for thetherapy of hematologic cancer or malignant disease states and for thediagnosis of hematologic cancer disease states. In one embodiment, thepresent invention provides a method of impairing a hematologic cancerprogenitor cell comprising contacting the cell with a compound thatselectively binds to CD123 in an amount effective to impair theprogenitor hematologic cancer cell. This contacting step may occur invarious environments, including in vitro and in vivo in the body of ananimal, including a human.

Throughout this application, reference will be made specifically toleukemia in describing certain embodiments of the present invention.However, it is understood that the present invention is not limited todiagnosis and treatment of leukemia or malignant lymphoproliferativedisorders alone, but to any disease in which the cancerous cellsselectively express CD123, which includes the genus of hematologiccancer.

In one embodiment, the present invention is directed to a method ofdetecting the presence of CD123 on, for example, a leukemia progenitorcell. Thus, the invention is also directed to a method of diagnosingleukemia. It is understood that by using a labeled ligand to bind toCD123, it is possible to detect the presence of leukemia progenitorcells. Thus, it is also possible to diagnose the likelihood of the onsetof leukemia in patients possessing such leukemic progenitor cellsexpressing CD123. The CD123 binding ligand may be an antibody to CD123,or it may be any of a variety of molecules that specifically bind toCD123. Furthermore, the label can be chosen from any of a variety ofmolecules, including, but not limited to, enzymatic compounds, ornon-enzymatic compounds that serve as a reporter of the presence of theligand which has bound to the CD123 molecule. Examples of such labelsinclude those that are, for example, radioactive, fluorescent,chemiluminescent or absorbant-based, or a combination of the foregoing.In one embodiment, an assay is provided for detecting the presence ofprogenitor leukemia cells in a sample by detecting the presence of CD123in the sample, which may be accomplished by introducing a compound thatselectively binds to CD123 and determining whether the compound binds toa component of the sample.

In another series of embodiments, the present invention also providescompounds or molecules which mimic (mimetics) the three-dimensionalstructure of part or all of the compounds such as peptides, antibodies,carbohydrates, lipids or nucleic acids that bind to CD123, and in thecase of antibodies, of the binding pockets of the antibodies, or of thecomplementarity determining regions (CDR's).

The present invention also provides pharmaceutical preparationscomprising a pharmaceutically acceptable carrier; and any one or more ofthe CD123 specific compounds and mimetics described above.

In another set of embodiments, the present invention provides a methodfor the treatment of leukemia, comprising administering to a humansubject or other animal in need of such treatment a therapeuticallyeffective amount of the compounds or their mimetic pharmaceuticalcompositions described above.

In still another set of embodiments, the present invention provides amethod of selectively purging leukemic stem cells from bone marrow.These stems cells may give rise to leukemia progenitor cells, or theymay be the progenitor cells, which may be impaired by the method of theinvention using various compounds or their mimetics and cytotoxic agentsthat may be contacted to either a bone marrow sample or injected into abone marrow of an individual, thereby destroying at least some of theleukemic stem cells in the bone marrow.

BRIEF DESCRIPTION OF THE DRAWINGS

The present invention will become more fully understood from thedetailed description given herein below, and the accompanying drawingswhich are given by way of illustration only, and thus are not limitingof the present invention, and wherein:

FIGS. 1A and 1B show CD123 expression on normal and leukemichematopoietic cells. Representative examples of CD123 labeling in cellsderived from normal bone marrow (panel A) or primary AML peripheralblood (panel B) are provided. The inset polygon in panel A (left dotplot) indicates the percentage of total bone marrow that is positive forCD123 expression (7%). The smaller rectangular gate indicates theproportion of total marrow strongly positive for CD123 (1%). The centerdot plot in panel A shows bone marrow that was enriched for CD34+ cellsby selection on an immunoaffinity column (see methods). The gatesindicate the total CD34+ population and the CD34+/CD38− population. Thehistograms indicate the CD123 labeling for the two gated populations.Panel B shows the same analysis for the primary AML specimen. M1 markersin the histograms indicate expression levels that are higher than 99% ofisotype control samples. For each specimen, 50,000-100,000 events wereanalyzed.

FIG. 2 shows CD expression on five primitive AML populations. Fiveprimary AML specimens were labeled with CD34, CD38, and CD123 andanalyzed by flow cytometry. The figure shows CD123 labeling for theCD34+/CD38− gated populations from each sample. The dark sectionsindicate CD123 staining and the light sections indicate parallellabeling with an isotype control antibody. M1 markers in the histogramsindicate expression levels that are higher than 99% of isotype controlsamples. For each specimen, 50,000-100,000 events were analyzed.

FIGS. 3A and 3B show engraftment of CD123+ AML cells in NOD/SCID mice.Sorted CD34+/CD123+ primary AML cells were transplanted into anirradiated NOD/SCID mouse. Six weeks post-transplant, bone marrow cellswere isolated and analyzed for the presence of human (CD45+) leukemiccells. Panel A shows the CD34 vs. CD45 profile of an engrafted specimenusing antibodies that are specific to human cells. Panel B shows theCD34 vs. CD123 profile of the CD45+ gated human cell population. Foreach sample, 50,000 events were analyzed.

FIG. 4 shows CD123 and CD131 immunoblot analysis of primary AMLspecimens. Five primary AML samples were derived from peripheral bloodand sorted to isolate the CD34+ population, thus insuring a virtuallypure leukemic sample. In addition, the leukemic cell line TF-1 (TF-1)and normal marrow CD34+ cells (normal CD34+) were included as controls.Lysates were made from each population, subject to denaturing PAGE andanalyzed by immunoblot with an anti-CD123 antibody (top panel) or ananti-CD131 antibody (bottom panel). The arrowhead at the left of eachpanel indicates the position of the IL-3Rα (CD123) and IL-3Rβ (CD131)chains, respectively.

FIGS. 5A, 5B and 5C show phosphorylation of signal transductioncomponents in response to IL-3. Three primary AML samples (Lanes 1-3)were derived from peripheral blood and sorted to isolate the CD34+population. Samples were treated with (+) or without (−) 20 ng/ml IL-3for 15 minutes, then lysed and subjected to PAGE. Each gel was thenelectro-blotted and membranes were probed with antibodies specific toeither total or phosphorylated protein for Akt (A), Stat5 (B), andMek-1(C). The lane labeled C on each blot is an antibody control and isderived from NIH 3T3 cells treated +/−50 ng/ml PDGF (A and C), or TF-1cells treated +/−25 ng/ml GM-SCF (B).

FIGS. 6A, 6B and 6C show CD123 expression in primary ALL. Flowcytometric analysis of CD123 expression on three independent primary ALL(acute lymphoid leukemia) specimens. CD123 labeling is shown by thefilled (dark) plots and controls are shown by open plots.

FIGS. 7A, 7B and 7C show CD123 expression in primary CML. Flowcytometric analysis of CD123 expression on three independent primary CML(chronic myelogenous leukemia) specimens. CD123 labeling is shown by thefilled (dark) plots and controls are shown by open plots.

DETAILED DESCRIPTION OF THE INVENTION Definitions

As used herein, the term “antibody” means an immunoglobulin molecule, ora fragment of an immunoglobulin molecule, having the ability tospecifically bind to a particular antigen. Antibodies are well known tothose of ordinary skill in the science of immunology. As used herein,the term “antibody” means not only intact antibody molecules but alsofragments of antibody molecules retaining antigen-binding ability. Suchfragments are also well known in the art and are regularly employed bothin vitro and in vivo. In particular, as used herein, the term “antibody”means not only intact immunoglobulin molecules but also the well-knownactive fragments F(ab′)₂, Fab, Fv, Fd, V_(H) and V_(L).

As used herein, the terms “bind” or “bind(s)” shall mean anyinteraction, whether via direct or indirect means, which affects thespecified receptor or receptor subunit.

As used herein, the terms “binds selectively to” shall mean that thecompound, composition, formulation, etc. does not significantly bindIL3R beta chain, but does bind IL3R alpha chain.

As used herein, the terms “CD123”, “IL3R subunit alpha” and “IL3Rα”shall be used interchangeably to mean an antigenic determinant that isdetectable in leukemia precursor cells as described herein, but notdetectable on normal cells as described herein.

As used herein, the term “compound” shall mean any purity of activeingredient, including formulations, compositions, naturally-occurringplants or animals, etc. The compound may include molecules that arenaturally occurring, such as proteins, nucleic acids, single strandednucleic acids, lipids, carbohydrates, and antibodies. However, syntheticversions of these naturally occurring molecules may be made, so long asthey bind CD123. The compounds may comprise more than one component. Forexample, a compound may be a monoclonal antibody attached to a toxin.Or, it may be a lipid attached to a label. The compounds may furthercomprise mimetics, and aptamers, but which all retain their specificityto CD123.

As used herein, the term “impair” shall mean any decrease infunctionality or activity (including growth or proliferative activity).

As used herein, the term “hematologic cancer” refers to a cancer of theblood, and includes leukemia and malignant lymphoproliferativedisorders, among others. “Leukemia” refers to a cancer of the blood, inwhich too many white blood cells that are ineffective in fightinginfection are made, thus crowding out the other parts that make up theblood, such as platelets and red blood cells. It is understood thatcases of leukemia are classified as acute or chronic. Cancer cells inacute leukemias are blocked at an immature stage. However, they continueto multiply. Consequently, there is a large accumulation ofnon-functional immature cells and the concomitant loss of functionalcells. Chronic leukemias progress more slowly, with cancer cellsdeveloping to full maturity. Furthermore, the white blood cells may bemyelogenous or lymphoid. Thus, certain forms of leukemia may be, by wayof example, acute lymphatic (or lymphoblastic) leukemia (ALL); acutemyelogenic leukemia (AML); chronic lymphocytic leukemia (CLL); orchronic myelogenic leukemia (CML); and myelodysplastic syndrome.“Malignant lymphoproliferative disorders” may refer to a lymphoma, suchas multiple myeloma, non-Hodgkin's lymphoma, Burkitt's lymphoma, andfollicular lymphoma (small cell and large cell), among others. Forpurposes of this invention, at least some of the hematologic cancercells are characterized by cells that express CD123. Also, for thepurposes of this application, whenever leukemia or malignantlymphoproliferative disorders are mentioned, the diagnostic andtreatment method of the invention applies generally to hematologiccancer.

As used herein, the term “introducing” shall mean any means of delivery,whether in vivo or in vitro, including simple contact.

As used herein, the term “mimetic” means a compound or molecule whichmimics the three-dimensional structures of a site on CD123 to which acompound may bind, or the compound may be a molecule that mimics amolecule that binds to CD123. In the case of an anti-CD123 antibodybinding site, or paratope, or active site, “mimetic” means a compoundthat mimics the three-dimensional structure of any combination of theantibody hypervariable loops or complementarity determining regions(CDR's).

As used herein, the term “mimic” means the three-dimensional placementof atoms of the mimetic such that similar ionic forces, covalent forces,van der Waal's or other forces, and similar charge complementarity, orelectrostatic complementarity, exist between the atoms of the mimeticand the atoms of the binding site of the compound such as a peptide oran antibody such that the mimetic has a similar binding affinity forCD123 as the parent compound and/or such that the mimetic has a similareffect on the function of CD123 in vitro or in vivo.

In the case of anti-CD123 antibodies, within the antigen-binding portionof an antibody, as is well-known in the art, there are complementaritydetermining regions (CDRs), which directly interact with the epitope ofthe antigen, and framework regions (FRs), which maintain the tertiarystructure of the paratope. In both the heavy chain Fd fragment and thelight chain of IgG immunoglobulins, there are four framework regions(FR1 through FR4) separated respectively by three complementaritydetermining regions (CDR1 through CDR3). The CDRs, and in particular theCDR3 regions, and more particularly the heavy chain CDR3, are largelyresponsible for antibody specificity.

As used herein, the term “normal” means any non-pathogenic ornon-pathology-related cells or conditions.

As used herein, the terms “primitive” and “progenitor” shall beinterchangeable. As used herein, with respect to polypeptides andantibodies, the term “substantially pure” means that the polypeptidesare substantially free of other substances with which they may be foundin nature or in vivo systems to an extent practical and appropriate fortheir intended use. In particular, the polypeptides are sufficientlypure and are sufficiently free from other biological constituents oftheir host cells so as to be useful in, for example, generatingantibodies, sequencing, or producing pharmaceutical preparations. Bytechniques well known in the art, substantially pure polypeptides may beproduced in light of the nucleic acid and amino acid sequences disclosedherein. Because a substantially purified polypeptide of the inventionmay be admixed with a pharmaceutically acceptable carrier in apharmaceutical preparation, the polypeptide may comprise only a smallpercentage by weight of the preparation. The polypeptide is nonethelesssubstantially pure in that it has been substantially separated from thesubstances with which it may be associated in living systems.

As used herein with respect to compounds or mimetics, the term“substantially pure” means that the compounds are substantially free ofother substances with which they may be found, in nature, in in vivosystems; or as a result of chemical or other synthesis, to an extentpractical and appropriate for their intended use. In particular, thecompounds are sufficiently pure and are sufficiently free from otherbiological constituents of their hosts cells, or chemical or physicalconstituents of their synthesis so as to be useful in, producingpharmaceutical preparations. By techniques well known in the art (U.S.Pat. No. 5,648,379; Colman, P. G. Protein Science 3: 1687-1696, 1994;Malby, et al., Structure 2: 733-746, 1994; McCoy et al., J. MolecularBiol. 268: 570-584, 1997), substantially pure compounds or mimetics, maybe designed. Because a substantially purified compound of the inventionmay be admixed with a pharmaceutically acceptable carrier in apharmaceutical preparation, the compound may comprise only a smallpercentage by weight of the preparation. The compound is nonethelesssubstantially pure in that it has been substantially separated from thesubstances with which it may be associated in living, chemical or othersystems.

Mimetics That Bind to CD123

Compounds that target CD123 can be found. Phage display libraries can beused to determine the DNA encoding the polypeptide that binds to CD123.The principles of this approach are disclosed in U.S. Pat. No.5,837,500, which is incorporated by reference herein in its entirety.Other non-peptide molecules that may bind to CD123 include nucleicacids, and liposomes. Carbohydrates may also be used to target CD123. Itis possible that the compound may not be a naturally occurringbiological molecule. Such chemicals may be made by combinatoriallibraries which are well known in the art, with the assay goal being thebinding of the chemical compound to CD123. Liposomes may ensconcecertain toxins or other cell-impairing substances or cell-imagingcompounds may be used to target CD123. Numerous variations andcombinations of compounds as targeting agents are contemplated by themethod of the invention, so long as CD123 is targeted, with theknowledge that leukemia detection and leukemia treatment is kept inmind.

Mimetics of Anti-CD123 Antibodies

It is also possible to use the anti-idiotype technology to isolate orscreen for compounds or mimetics which mimic an epitope. Thus, ananti-idiotypic monoclonal antibody which is the image of the epitopebound by the first monoclonal antibody, since it effectively acts as anantigen, may be used to isolate mimetics from a combinatorial chemical,or other libraries, of chemical or other compounds, such as peptidephage display libraries (Scott and Smith, Science 249: 386-390, 1990;Scott and Craig, Curr. Opin. Biotechnol. 5: 40-48, 1992; Bonnycastle etal., J. Mol. Biol. 258: 747-762, 1996). Hence, peptides or constrainedpeptides mimicking proteins or other compounds, including those withnucleic acid, lipid, carbohydrate or other moieties, may be cloned(Harris et al., Proc. Natl. Acad. Sci. (USA) 94: 2454-2459, 1997).

Purely synthetic molecules, which may not occur in nature and aretherefore more resistant to catabolism, excretion or degradation, may bedesigned by the three-dimensional placement of atoms, such that similarionic forces, covalent forces, van der Waal's or other forces, andsimilar charge complementarity, or electrostatic complementarity, existbetween the atoms of the mimetic and the atoms of the antigenic bindingsite or epitope. These mimetics may then be screened for high affinitybinding to CD123 and detect and/or impair the CD123 bearing cell invitro or in vivo, as described in more detail below.

Diagnostic and Pharmaceutical Preparations

The invention also relates to a method for preparing diagnostic orpharmaceutical compositions comprising the CD123 binding compound andits mimetics. The pharmaceutical preparation includes a pharmaceuticallyacceptable carrier. Such carriers, as used herein, mean non-toxicmaterials that do not interfere with the effectiveness of the biologicalactivity of the active ingredients. The term “physiologicallyacceptable” refers to a non-toxic material that is compatible with abiological system such as a cell, cell culture, tissue, or organism. Thecharacteristics of the carrier will depend on the route ofadministration. Physiologically and pharmaceutically acceptable carriersinclude diluents, fillers, salts, buffers, stabilizers, solubilizers,and other materials that are well known in the art.

The anti-CD123 antibodies and mimetics may be labeled by a variety ofmeans for use in diagnostic and/or pharmaceutical applications. Thereare many different labels and methods of labeling known to those ofordinary skill in the art. Examples of the types of labels which can beused in the present invention include enzymes, radioisotopes,fluorescent compounds, colloidal metals, chemiluminescent compounds, andbioluminescent compounds. Those of ordinary skill in the art will knowof other suitable labels for binding to the CD123 binding compound, suchas monoclonal antibodies, or mimetics thereof, or will be able toascertain such, using routine experimentation. Furthermore, the bindingof these labels to the CD123 specific compounds or their mimetics can bedone using standard techniques common to those of ordinary skill in theart.

In the case of antibodies, another labeling technique which may resultin greater sensitivity consists of coupling the antibodies or mimeticsto low molecular weight haptens. These haptens can then be specificallyaltered by means of a second reaction. For example, it is common to usehaptens such as biotin, which reacts with avidin, or dinitrophenol,pyridoxal, or fluorescein, which can react with specific anti-haptenantibodies.

Diagnostic and Treatment Kits

The materials for use in the assay of the invention are ideally suitedfor the preparation of a kit. Such a kit may comprise a carrier meansbeing compartmentalized to receive in close confinement one or morecontainer means such as vials, tubes, and the like, each of thecontainer means comprising one of the separate elements to be used inthe method. For example, one of the container means may comprise acompound that binds to CD123, such as a monoclonal antibody, or amimetic thereof, which is, or can be, detectably labeled with a labelthat is suitable for diagnostic purposes or if treatment is desired, acytotoxic or impairing agent. In the case of a diagnostic kit, the kitmay also have containers containing buffer(s) and/or a containercomprising a reporter-means, such as a biotin-binding protein, such asavidin or streptavidin, bound to a reporter molecule, such as anenzymatic or fluorescent label. In addition to the chemical material, ofcourse a means of instructions for using the kit is included, preferablyfor either diagnosing leukemia, or treating leukemia. The instructionmeans may be written on the vial, tube and the like, or written on aseparate paper, or on the outside or inside of the container. Theinstructions may also be in the form of a multi-media format, such asCD, computer disk, video and so on.

Preparation of Immunotoxins

While the preparation of immunotoxins is, in general, well known in theart (see, e.g., U.S. Pat. No. 4,340,535, and EP 44167, both incorporatedherein by reference), the inventors are aware that certain advantagesmay be achieved through the application of certain preferred technology,both in the preparation of the immunotoxins and in their purificationfor subsequent clinical administration. For example, while IgG basedimmunotoxins will typically exhibit better binding capability and slowerblood clearance than their Fab′ counterparts, Fab′ fragment-basedimmunotoxins will generally exhibit better tissue penetrating capabilityas compared to IgG based immunotoxins.

Additionally, while numerous types of disulfide-bond containing linkersare known which can successfully be employed to conjugate the toxinmoiety with the binding agent, certain linkers will generally bepreferred over other linkers, based on differing pharmacologiccharacteristics and capabilities. For example, linkers that contain adisulfide bond that is sterically “hindered” are to be preferred, due totheir greater stability in vivo, thus preventing release of the toxinmoiety prior to binding at the site of action.

Cross-linking reagents are used to form molecular bridges that tietogether functional groups of two different proteins (e.g., a toxin anda binding agent). To link two different proteins in a step-wise manner,hetero bifunctional cross-linkers can be used which eliminate theunwanted homopolymer formation. An exemplary hetero bifunctionalcross-linker contains two reactive groups: one reacting with primaryamine group (e.g., N-hydroxy succinimide) and the other reacting with athiol group (e.g., pyridyl disulfide, maleimides, halogens, etc.).Through the primary amine reactive group, the cross-linker may reactwith the lysine residue(s) of one protein (e.g., the selected antibodyor fragment) and through the thiol reactive group, the cross-linker,already tied up to the first protein, reacts with the cysteine residue(free sulfhydryl group) of the other protein (e.g., dgA).

The spacer arm between these two reactive, groups of any cross-linkersmay have various lengths and chemical compositions. A longer spacer armallows a better flexibility of the conjugate components while someparticular components in the bridge (e.g., benzene group) may lend extrastability to the reactive group or an increased resistance of thechemical link to the action of various aspects (e.g., disulfide bondresistant to reducing agents).

The most preferred cross-linking reagent is SMPT, which is abifunctional cross-linker containing a disulfide bond that is“sterically hindered” by an adjacent benzene ring and methyl groups. Itis believed that stearic hindrance of the disulfide bond serves afunction of protecting the bond from attack by thiolate anions such asglutathione which can be present in tissues and blood, and thereby helpin preventing decoupling of the conjugate prior to its delivery to thesite of action by the binding agent. The SMPT cross-linking reagent, aswith many other known cross-linking reagents, lends the ability tocross-link functional groups such as the SH of cysteine or primaryamines (e.g., the epsilon amino group of lysine). Another possible typeof cross-linker includes the hetero-bifunctional photoreactivephenylazides containing a cleavable disulfide bond such assulfosuccinimidyl-2-(p-azido salicylamido)ethyl-1,3′-dithiopropionate.The N-hydroxy-succinimidyl group reacts with primary amino groups andthe phenylazide (upon photolysis) reacts non-selectively with any aminoacid residue.

Although the “hindered” cross-linkers will generally be preferred in thepractice of the invention, non-hindered linkers can be employed andadvantages in accordance herewith nevertheless realized. Other usefulcross-linkers, not considered to contain or generate a protecteddisulfide, include SATA, SPDP and 2-iminothiolane. The use of suchcross-linkers is well understood in the art.

Once conjugated, it will be important to purify the conjugate so as toremove contaminants such as unconjugated A chain or binding agent. It isimportant to remove unconjugated A chain because of the possibility ofincreased toxicity. Moreover, it is important to remove unconjugatedbinding agent to avoid the possibility of competition for the antigenbetween conjugated and unconjugated species. In any event, a number ofpurification techniques are disclosed in the Examples below which havebeen found to provide conjugates to a sufficient degree of purity torender them clinically useful. In general, the most preferred techniquewill incorporate the use of Blue-Sepharose with a gel filtration or gelpermeation step. Blue-Sepharose is a column matrix composed of CibacronBlue. 3GA and agarose, which has been found to be useful in thepurification of immunoconjugates. The use of Blue-Sepharose combines theproperties of ion exchange with A chain binding to provide goodseparation of conjugated from unconjugated binding.

The Blue-Sepharose allows the elimination of the free (non conjugated)binding agent (e.g., the antibody or fragment) from the conjugatepreparation. To eliminate the free (unconjugated) toxin (e.g., dgA) amolecular exclusion chromatography step is preferred using eitherconventional gel filtration procedure or high performance liquidchromatography.

After a sufficiently purified conjugate has been prepared, one willdesire to prepare it into a pharmaceutical composition that may beadministered parenterally. This is done by using for the lastpurification step a medium with a suitable pharmaceutical composition.

Suitable pharmaceutical compositions in accordance with the inventionwill generally comprise from about 10 to about 100 mg of the desiredconjugate admixed with an acceptable pharmaceutical diluent orexcipient, such as a sterile aqueous solution, to give a finalconcentration of about 0.25 to about 2.5 mg/ml with respect to theconjugate. Such formulations will typically include buffers such asphosphate buffered saline (PBS), or additional additives such aspharmaceutical excipients, stabilizing agents such as BSA or HSA, orsalts such as sodium chloride. For parenteral administration it isgenerally desirable to further render such compositions pharmaceuticallyacceptable by insuring their sterility, non-immunogenicity andnon-pyrogenicity. Such techniques are generally well known in the art asexemplified by Remington's Pharmaceutical Sciences, 16th Ed. MackPublishing Company, 1980, incorporated herein by reference. It should beappreciated that endotoxin contamination should be kept minimally at asafe level, for example, less than 0.5 ng/mg protein. Moreover, forhuman administration, preparations should meet sterility, pyrogenicity,general safety and purity standards as required by FDA Office ofBiological Standards.

A preferred parenteral formulation of the immunotoxins in accordancewith the present invention is 0.25 to 2.5 mg conjugate/ml in 0.15M NaClaqueous solution at pH 7.5 to 9.0. The preparations may be stored frozenat −10° C. to −70° C. for at least 1 year.

It is contemplated that most therapeutic applications of the presentinvention will involve the targeting of a toxin moiety (cytotoxic agent)to the CD123 leukemia marker. This is due to the much greater ability ofmost toxins to deliver a cell killing effect as compared to otherpotential agents.

However, there may be circumstances such as when the target antigen doesnot internalize by a route consistent with efficient intoxication byimmunotoxins, where one will desire to target chemotherapeutic agentssuch as cytokines, antimetabolites, alkylating agents, hormones, and thelike. The advantages of these agents over their non-antibody conjugatedcounterparts is the added selectivity afforded by the antibody. Onemight mention by way of example agents such as steroids, cytosinearabinoside, methotrexate, aminopterin, anthracyclines, mitomycin C,vinca alkaloids, demecolcine, etopside, mithramycin, and the like. Thislist is, of course, merely exemplary in that the technology forattaching pharmaceutical agents to antibodies for specific delivery totissues is well established.

One preferred cytotoxic moiety for use in the present invention is aradioisotope, which can be coupled to or conjugated with, for example,an anti-CD123 antibody. Preferred radioisotopes include α-emitters suchas, for example, ²¹¹Astatine, ²¹²Bismuth and ²¹³Bismuth, as well asβ-emitters such as, for example, ¹³¹Iodine, ⁹⁰Yttrium, ¹⁷⁷Lutetium,¹⁵³Samarium and ¹⁰⁹Palladium. Particularly preferred radioisotopes are²¹¹Astatine and ¹³¹Iodine.

It is proposed that particular benefits may also be achieved through theapplication of the invention to cell imaging. Imaging of leukemia cellsis believed to provide a major advantage when compared to availableimaging techniques, in that the cells are readily accessible.

Moreover, the technology for attaching paramagnetic, radioactive andeven fluorogenic ions to antibodies is well established. Many of thesemethods involve the use of a metal chelate complex employing, forexample, an organic chelating agent such a DTPA attached to the antibody(see, e.g., U.S. Pat. No. 4,472,509). In the context of the presentinvention the selected ion is thus targeted to the cancerous area by theantibody, allowing imaging to proceed by means of the attached ion.

In a preferred embodiment, in the method of the invention, theantibodies may also be fused to a protein effector molecule byrecombinant means such as through the use of recombinant DNA techniquesto produce a nucleic acid which encodes both the antibody and theeffector molecule and expressing the DNA sequence in a host cell such asE. coli. The DNA encoding the chimeric protein may be cloned in cDNA orin genomic form by any cloning procedure known to those skilled in theart. See for example Sambrook et al., Molecular Cloning: A LaboratoryManual, Cold Spring Harbor Laboratory, (1989), which is hereinincorporated by reference.

Fusion or conjugation of antibodies to various labels produces a highlyspecific detectable marker that may be used to detect the presence orabsence of cells or tissues bearing the particular molecule to which theantibody is detected. Alternatively, the antibodies may be chemicallyconjugated or fused to an effector molecule that is another specificbinding moiety, e.g. a ligand such as those described above. In thisform the composition will act as a highly specific bifunctional linker.This linker may act to bind and enhance the interaction between cells orcellular components to which the fusion protein binds. Thus, forexample, where the fusion protein is a growth factor joined to anantibody or antibody fragment (e.g. an Fv fragment of an antibody), theantibody may specifically bind antigen positive cancer cells while thegrowth factor binds receptors on the surface of immune cells. The fusionprotein may thus act to enhance and direct an immune response towardtarget cancer cells.

In Vitro Detection and Diagnostics

The method of using the compounds that bind to CD123 and their mimeticsare suited for in vitro use, for example, in immunoassays in which theycan be utilized in liquid phase or bound to a solid phase carrier. Inaddition, the monoclonal antibodies in these immunoassays can bedetectably labeled in various ways. Examples of types of immunoassayswhich can utilize the monoclonal antibodies and their mimetics arecompetitive and non-competitive immunoassays in either a direct orindirect format. Examples of such immunoassays are the radioimmunoassay(RIA) and the sandwich (immunometric) assay. Detection of antigens usingthe monoclonal antibodies and their mimetics can be done utilizingimmunoassays which are run in either the forward, reverse, orsimultaneous modes, including immunohistochemical assays onphysiological samples. Those of skill in the art will know, or canreadily discern, other immunoassay formats without undueexperimentation.

The compounds that bind CD123 and mimetics can be bound to manydifferent carriers and used to detect the presence of CD123 bearingleukemia cells, including progenitor cells. Examples of well-knowncarriers include glass, polystyrene, polypropylene, polyethylene,dextran, nylon, amylase, natural and modified cellulose, polyacrylamide,agarose and magnetite. The nature of the carrier can be either solubleor insoluble for purposes of the invention. Those skilled in the artwill know of other suitable carriers for binding various compounds, orwill be able to ascertain such, using routine experimentation.

For purposes of the invention, CD123 may be detected by the compoundsand their mimetics when present in biological fluids and tissues. Anysample containing a detectable amount of CD123 ectopeptide can be used.A sample can be a liquid such as urine, saliva, cerebrospinal fluid,blood, serum or the like; a solid or semi-solid such as tissues, feces,or the like; or, alternatively, a solid tissue such as those commonlyused in histological diagnosis.

In Vivo Detection of CD123

In using the CD123 binding compounds and mimetics for the in vivodetection of CD123, the detectably labeled compound or its mimetic isgiven in a dose which is diagnostically effective. The term“diagnostically effective” means that the amount of detectably labeledcompound, such as monoclonal antibody or mimetic is administered insufficient quantity to enable detection of the leukemia cells for whichthe compounds or mimetics are specific.

The concentration of detectably labeled compound or mimetic which isadministered should be sufficient such that the binding to CD123 orCD123-bearing leukemia cells, is detectable compared to the background.Further, it is desirable that the detectably labeled compound or mimeticbe rapidly cleared from the circulatory system in order to give the besttarget-to-background signal ratio.

As a rule, the dosage of detectably labeled compound or mimetic for invivo diagnosis will vary depending on such factors as age, sex, andextent of disease of the individual. The dosage of the compound can varyfrom about 0.01 mg/kg to about 500 mg/kg, preferably about 0.1 mg/kg toabout 200 mg/kg, most preferably about 0.1 mg/kg to about 10 mg/kg. Suchdosages may vary, for example, depending on whether multiple injectionsare given, on the tissue being assayed, and other factors known to thoseof skill in the art.

For in vivo diagnostic imaging, the type of detection instrumentavailable is a major factor in selecting an appropriate radioisotope.The radioisotope chosen must have a type of decay which is detectablefor the given type of instrument. Still another important factor inselecting a radioisotope for in vivo diagnosis is that the half-life ofthe radioisotope be long enough such that it is still detectable at thetime of maximum uptake by the target, but short enough such thatdeleterious radiation with respect to the host is acceptable. Ideally, aradioisotope used for in vivo imaging will lack a particle emission butproduce a large number of photons in the 140-250 keV range, which may bereadily detected by conventional gamma cameras.

For in vivo diagnosis, radioisotopes may be bound to the compound eitherdirectly or indirectly by using an intermediate functional group.Intermediate functional groups which often are used to bindradioisotopes which exist as metallic ions are the bifunctionalchelating agents such as diethylenetriaminepentacetic acid (DTPA) andethylenediaminetetra-acetic acid (EDTA) and similar molecules. Typicalexamples of metallic ions which can be bound to the monoclonalantibodies and mimetics of the invention are ¹¹¹In, ⁹⁷Ru, ⁶⁷Ga, ⁶⁸Ga,⁷²As, ⁸⁹Zr, ^(99m)Tc, ¹²³I and ²⁰¹Tl.

In the diagnosis method of the invention, the compounds and mimetics canalso be labeled with a paramagnetic isotope for purposes of in vivodiagnosis, as in magnetic resonance imaging (MRI) or electron spinresonance (ESR). In general, any conventional method for visualizingdiagnostic imaging can be utilized. Usually gamma and positron emittingradioisotopes are used for camera imaging and paramagnetic isotopes forMRI. Elements which are particularly useful in such techniques include¹⁵⁷Gd, ⁵⁵Mn, ¹⁶²Dy, ⁵²Cr and ⁵⁶Fe.

In the cell monitoring method of the invention, the compounds andmimetics can be used in vitro and in vivo to monitor the course ofleukemia disease therapy. Thus, for example, by measuring the increaseor decrease in the biological molecules associated with such a diseasesor changes in the concentration of CD123 ectopeptide or CD123 bearingleukemia cells present in the body or in various body fluids, it wouldbe possible to determine whether a particular therapeutic regimen aimedat ameliorating the above leukemia disease is effective.

Prophylaxis and Therapy of Leukemia

The CD123 specific compounds can also be used therapeutically fortreatment of leukemia in both humans and other animals. The term,“therapeutically” or “therapy” as used herein in conjunction with themethod of the invention is directed to using CD123 binding compounds,such as anti-CD123 monoclonal antibodies and their mimetics, whichdenotes both prophylactic as well as therapeutic administration and bothpassive immunization with substantially purified polypeptide products,and mimetics, as well as gene therapy by transfer of polynucleotidesequences encoding the product or part thereof. Thus, the compounds andmimetics can be administered to high-risk subjects in order to lessenthe likelihood and/or severity of leukemia relapse, or administered tosubjects already evidencing active leukemia disease.

For certain applications, it is envisioned that pharmacologic agentswill serve as useful agents for attachment to the compounds,particularly cytotoxic or otherwise anticellular agents having theability to kill or suppress the growth or cell division of leukemiacells. In general, the invention contemplates the use of anypharmacologic agent that can be conjugated to a CD123 binding compoundand delivered in active form to the targeted cell. Exemplaryanticellular agents include chemotherapeutic agents, radioisotopes aswell as cytotoxins. In the case of chemotherapeutic agents, theinventors propose that agents such as a hormone such as a steroid; anantimetabolite such as cytosine arabinoside, fluorouracil, methotrexateor aminopterin; an anthracycline; mitomycin C; a vinca alkaloid;demecolcine; etoposide; mithramycin; calicheamicin, CC-1065 andderivatives thereof, or an alkylating agent such as chlorambucil ormelphalan, will be particularly preferred. Other embodiments may includeagents such as a coagulant, a cytokine, growth factor, bacterialendotoxin or the lipid A moiety of bacterial endotoxin. In any event, itis proposed that agents such as these may be successfully conjugated toantibodies in a manner that will allow their targeting, internalization,release or presentation to blood components at the site of the targetedleukemia cells as required using known conjugation technology.

In certain preferred embodiments, cytotoxic agents for therapeuticapplication will include generally a plant-, fungus- or bacteria-derivedtoxin, such as an A chain toxins, a ribosome inactivating protein,α-sarcin, aspergillin, restirictocin, a ribonuclease, diphtheria toxinor pseudomonas exotoxin, to mention just a few examples. The use oftoxin-antibody constructs is well known in the art of immunotoxins, asis their attachment to antibodies. Of these, a particularly preferredtoxin for attachment to antibodies will be a deglycosylated ricin Achain. Deglycosylated ricin A chain is preferred because of its extremepotency, longer half-life, and because it is economically feasible tomanufacture a clinical grade and scale.

In other preferred embodiments, the cytotoxic agent may be aradioisotope. Preferred radioisotopes include α-emitters such as, forexample, ²¹¹Astatine, ²¹²Bismuth and ²¹³Bismuth, as well as β-emitterssuch as, for example, ¹³¹Iodine, ⁹⁰Yttrium, ¹⁷⁷Lutetium, ¹⁵³Samarium and¹⁰⁹Palladium.

As used herein, a “therapeutically effective amount” of a compound is adosage large enough to produce the desired effect in which the symptomsof leukemia or the likelihood of onset of leukemia is decreased. Atherapeutically effective amount is not, however, a dosage so large asto cause adverse side effects, such as hyperviscosity syndromes,pulmonary edema, congestive heart failure, and the like. Generally, atherapeutically effective amount may vary with the subject's age,condition, and sex, as well as the extent of the disease in the subjectand can be determined by one of skill in the art. The dosage may beadjusted by the individual physician or veterinarian in the event of anycomplication. A therapeutically effective amount may vary from about0.01 mg/kg to about 500 mg/kg, preferably from about 0.1 mg/kg to about200 mg/kg, most preferably from about 0.2 mg/kg to about 20 mg/kg, inone or more dose administrations daily, for one or several days.

In the method of the invention, the compounds and their mimetics can beadministered by injection or by gradual infusion over time. Theadministration of the compounds and their mimetics may be, for example,intravenous, intraperitoneal, intramuscular, intracavity, subcutaneous,or transdermal.

Preparations for parenteral administration include sterile aqueous ornon-aqueous solutions. suspensions, and emulsions. Examples ofnon-aqueous solvents are propylene glycol, polyethylene glycol,vegetable oils such as olive oil, and injectable organic esters such asethyl oleate. Aqueous carriers include water, alcoholic/aqueoussolutions, emulsions or suspensions, including saline and bufferedmedia. Parenteral vehicles include sodium chloride solution, Ringer'sdextrose, dextrose and sodium chloride, lactated Ringer's or fixed oils.Intravenous vehicles include fluid and nutrient replenishers,electrolyte replenishers (such as those based on Ringer's dextrose), andthe like. Preservatives and other additives may also be present such as,for example, antimicrobials, anti-oxidants, chelating agents, and inertgases and the like.

Depending on the specific clinical status of the disease, administrationcan be made via any accepted systemic delivery system, for example, viaoral route or parenteral route such as intravenous, intramuscular,subcutaneous or percutaneous route, or vaginal, ocular or nasal route,in solid, semi-solid or liquid dosage forms, such as for example,tablets, suppositories, pills, capsules, powders, solutions,suspensions, cream, gel, implant, patch, pessary, aerosols, collyrium,emulsions or the like, preferably in unit dosage forms suitable for easyadministration of fixed dosages. The pharmaceutical compositions willinclude a conventional carrier or vehicle and a CD123 binding compoundand, in addition, may include other medicinal agents, pharmaceuticalagents, carriers, adjuvants, and so on.

If desired, the pharmaceutical composition to be administered may alsocontain minor amounts of non-toxic auxiliary substances such as wettingor emulsifying agents, pH buffering agents and the like, such as forexample, sodium acetate, sorbitan monolaurate, triethanolamine oleate,and so on.

The compounds of this invention are generally administered as apharmaceutical composition which comprises a pharmaceutical vehicle incombination with a CD123 binding compound. The amount of the drug in aformulation can vary within the full range employed by those skilled inthe art, e.g., from about 0.01 weight percent (wt %) to about 99.99 wt %of the drug based on the total formulation and about 0.01 wt % to 99.99wt % excipient.

The preferred mode of administration, for the conditions mentionedabove, is oral administration using a convenient daily dosage regimenwhich can be adjusted according to the degree of the complaint. For saidoral administration, a pharmaceutically acceptable, non-toxiccomposition is formed by the incorporation of the selected CD123 bindingcompound in any of the currently used excipients, such as, for example,pharmaceutical grades of mannitol, lactose, starch, magnesium stearate,sodium saccharine, talc, cellulose, glucose, gelatin, sucrose, magnesiumcarbonate, and the like. Such compositions take the form of solutions,suspensions, tablets, pills, capsules, powders, sustained releaseformulations and the like. Such compositions may contain between about0.01 wt % and 99.99 wt % of the active compound according to thisinvention.

Preferably the compositions will have the form of a sugar coated pill ortablet and thus they will contain, along with the active ingredient, adiluent such as lactose, sucrose, dicalcium phosphate, and the like; adisintegrant such as starch or derivatives thereof; a lubricant such asmagnesium stearate and the like; and a binder such as starch,polyvinylpyrrolidone, acacia gum, gelatin, cellulose and derivativesthereof, and the like.

It is understood that by “pharmaceutical composition”, it is meant thatthe CD123 binding compound is formulated into a substance that is to beadministered purposefully diagnosing or treating leukemia in theindividual. And, by “pharmaceutical composition”, it excludes thosecompositions that are used to administer to individuals as testcompounds for a purpose other than as a diagnostic or treatment agentfor leukemia.

The invention is described in further detail hereinbelow.

Several recent studies have suggested the presence and importance ofstem cells in both the genesis and perpetuation of AML. Phenotypically,cells described as CD34+/CD38− or CD34+/HLA-DR− appear to play a centralrole in the development of leukemic populations (Bonnet D, et al., Humanacute myeloid leukemia is organized as a hierarchy that originates froma primitive hematopoietic cell. Nat. Med. 1997, 3: 730-737; Blair A, etal., Most acute myeloid leukemia progenitor cells with long-termproliferative ability in vitro and in vivo have the phenotypeCD34(+)/CD71(−)/HLA-DR−. Blood 1998, 92: 4325-35). Furthermore, there isevidence suggesting that such cells may be relatively resistant tochemotherapeutic drugs, and consequently contribute to the phenomenon ofrelapse (Terpstra W, et al., Fluorouracil selectively spares acutemyeloid leukemia cells with long-term growth abilities inimmunodeficient mice and in culture. Blood 1996, 88: 1944-50). Thus, abetter understanding of LSC biology and the characterization of uniqueLSC antigens are essential to the development of better treatments forAML.

While the various AML subtypes display considerable diversity withrespect to developmental characteristics, phenotype, cytokineresponsiveness, etc., there appears to be a marked degree of functionalconservation at the level of more primitive leukemic cells. This featurehas been demonstrated by the work of Bonnet et. al., in which aCD34+/CD38− subpopulation was shown to be sufficient to establishleukemia in NOD/SCID mice (Bonnet D, et al., Human acute myeloidleukemia is organized as a hierarchy that originates from a primitivehematopoietic cell. Nat. Med. 1997, 3: 730-737). Similar studies byothers have corroborated the existence of leukemic stem cells for bothAML and CML and confirmed their relatively homogeneous phenotype andfunctional capacity (Blair A, Hogge et al., Most acute myeloid leukemiaprogenitor cells with long-term proliferative ability in vitro and invivo have the phenotype CD34(+)/CD71(−)/HLA-DR−. Blood 1998, 92:4325-35; Holyoake T, et al., Isolation of a highly quiescentsubpopulation of primitive leukemic cells in chronic myeloid leukemia.Blood 1999, 94: 2056-64). However, to date, no study has identified anantigenic feature of myeloid LSC's that may allow their identificationor preferential targeting for ablative therapy. In this report, we haveidentified an additional commonality among CD34+/CD38− AML stem cells,expression of CD123, which facilitates their discrimination from normalhematopoietic stem cells. While the CD123 antigen was readily detectedat high levels on AML cells, the IL-3 receptor β chain, CD131, was notdetected.

Our experiments indicate that the transcription factor IRF-1 (Interferonregulatory factor-1) is over-expressed (in 6 of 6 primary AML specimensexamined). Previous studies by Korpelainen et. al. have shown thattreatment of endothelial cells with IFN-γ results in up-regulation ofCD123 (Korpelainen E I, et al., Interferon-gamma upregulatesinterleukin-3 (IL-3) receptor expression in human endothelial cells andsynergizes with IL-3 in stimulating major histocompatibility complexclass II expression and cytokine production. Blood 1995, 86: 176-82).Similarly, our own studies have shown that treatment of primary AMLcells with IFN-γ increases expression of CD123 (data not shown). Thus,aberrant expression of interferon regulatory molecules might play a rolein controlling CD123 expression in AML cells.

Expression of the CD123 antigen formally demonstrates that LSC's arebiologically distinct from their normal stem cell counterparts. BecauseCD123 is not readily found on normal hematopoietic stem cells, itprovides a unique marker that can be used to identify malignant tissue.This feature may be useful for research purposes, as well as in minimalresidual disease (MRD) studies. Further, the CD123 epitope represents atarget to which therapeutic strategies may be directed. Previousclinical trials have used monoclonal antibodies against both the CD33and CD45 antigens as a means to deliver radioisotopes to AML cells invivo (Appelbaum F R., Antibody-targeted therapy for myeloid leukemia.Semin Hematol 1999, 36: 2-8.). In addition, several other recent studieshave shown exciting results using monoclonal antibodies specific toantigens on malignant cells such as CD20, CD52, and Her-2 (Maloney D G.,Advances in immunotherapy of hematologic malignancies. Curr Opin Hematol1998; 5: 237-43; Sikic B I., New approaches in cancer treatment. AnnOncol 1999, 10 Suppl 6: 149-53). Antibodies to CD123 may be useful in asimilar paradigm and will be capable of delivering a cytotoxic hit thatspecifically targets the leukemic stem cell population.

We have shown that CD123 represents a unique antigenic marker for theidentification of primitive leukemic cells from a broad range of humanspecimens across a broad range of leukemic diseases. Our studies showthat CD123 is generally expressed at high levels and may be indicativeof previously uncharacterized aspects of leukemia biology.

The present invention will be further illustrated in the following,non-limiting Examples. The Examples are illustrative only and do notlimit the claimed invention regarding the materials, conditions, processparameters and the like recited herein.

EXAMPLES Example 1 Materials and Methods: Cell Processing

Primary AML cells were obtained from the peripheral blood or bone marrowof patients. Normal bone marrow was obtained as waste material followingpathological analysis, surgical marrow harvest, or from the NationalDisease Research Interchange (NDRI). Marrow cells were depleted oferythrocytes by suspending in 150 mM NH₄Cl+10 mM NaHCO₃ for 5 minutes,followed by two washes with phosphate buffered saline (PBS). Blood cellswere subjected to Ficoll-Paque (Pharmacia) density gradient separationto isolate the mononuclear white blood cell compartment. Resultingleukocytes from marrow or blood were then used for immunoaffinityselection, and/or flow cytometric analysis or sorting. For CD34+ cellselection, the Miltenyi immunoaffinity device (varioMACS) was usedaccording to the manufacturer's instructions. In some cases, leukocyteswere cryopreserved at a concentration of 5×10⁷ cells/ml in freezingmedium consisting of Iscoves modified Dulbecco medium (IMDM), 40% fetalbovine serum (FBS) and 10% dimethylsulfoxide (DMSO).

Example 2 Flow Cytometry

Cytokine receptors were detected by labeling with the followingmonoclonal antibodies: CD114-biotin, CD116-FITC, CD123-PE, CD131-biotin(all from Pharmingen), CD117-PE (Coulter), and CD135-PE (Caltag).Biotinylated antibodies were visualized by subsequent labeling withstreptavidin-PE (SA-PE, Becton Dickinson). Primitive AML subpopulationswere identified using CD34-FITC or CD34-PE in combination with CD38-APC(Becton Dickinson). Primary AML cells were identified in NOD/SCID miceusing CD45-PE (Pharmingen) specific to human cells. To analyze cellstransplanted into NOD/SCID mice, bone marrow was harvested at 6-8 weekspost-transplantation. Cells were blocked with the anti-Fc receptorantibody 2.4G2 and 25% human serum, followed by double-labeling withhuman-specific CD34-FITC and CD45-PE antibodies. Control samplesconsisted of marrow cells from non-transplanted mice. In some cases,cells were also labeled with CD123-PE to ensure sustained expression ofthe CD123 antigen. For each specimen 50,000-100,000 events wereanalyzed. Using this approach, human cells could reliably be detected toa frequency as low as 0.1%. Any analysis falling below 0.1% positivecells was considered negative.

Example 3 Immunoblots

Cell samples were lysed at a concentration of 2×10⁷ cells/ml in PBScontaining: 1% NP-40, 0.5% deoxycholate, 0.1% sodium dodecyl sulfate(SDS), 1 mM sodium vanadate (Na₃VO₄), 30 μl aprotinin (Sigma), 1 mMphenylmethylsulfonyl fluoride (Sigma), 1 μg/ml pepstatin, and 1 μg/mlleupeptin (Oncogene Research); incubated on ice for 30 minutes, andcentrifuged at 15,000×g for 10 minutes to remove debris. The resultingprotein lysate was then aliquoted and stored at −80° C. For immunoblotanalysis, protein lysates were thawed and mixed with sample buffer andreducing agent (Novex, San Diego, Calif., per manufacturer'sinstructions), and heated at 70° C. for 10 minutes. Samples were thenimmediately analyzed by denaturing PAGE (Novex, 4-12% Bis-Tris or 7%Tris-Acetate gels) using the equivalent of 4×10⁵ cells per lane.Following electrophoresis, samples were electro-transferred ontoImmobilon-P membrane (Millipore) and probed with the indicatedantibodies. To detect CD123 (IL-3R alpha chain), antibodies S-12 (SantaCruz Biotech) or 9F5 (Pharmingen) were used. For the analysis of Mek andAkt, protein-specific and phosphoprotein-specific rabbit polyclonalantibodies from New England Biolabs were used. Anti-Stat5 polyclonal(Transduction Labs) and anti-phospho-Stat5 (New England Biolabs) wereused to analyze the phosphorylation status of Stat5. All primaryantibodies were detected using alkaline phosphatase-conjugated secondaryantibodies (Santa Cruz Biotechnology) and the ECF reagent (PharmaciaBiotech) per manufacturer's instructions. Blots were visualized using aMolecular Dynamics STORM 860 system and Imagequant™ Software.

Example 4 NOD/SCID Mouse Assays

NOD/SCID mice (Jackson Laboratories, Bar Harbor, Me.) were exposed to225 rads of γ-irradiation from a ¹³⁷Cs source. Cells to be assayed wereresuspended in 0.25 mls HBSS (Hanks balanced salt solution, Gibco) with2% FBS and injected IV into the tail vein. For the analysis of somesorted populations, 1×10⁶ irradiated (2500 Rads) mouse bone marrow cellswere co-injected as carrier. After 6-8 weeks, animals were sacrificedand bone marrow was analyzed for the presence of human cells using flowcytometry (see above).

Example 5 Results

Analysis of cytokine receptors demonstrates strong expression of CD123on primitive leukemic but not normal cells. Multiparameter flowcytometry was used to analyze the expression of cytokine receptorspreviously implicated in the growth of malignant hematopoietic cells.While several receptors displayed interesting patterns of antibodylabeling, the most striking feature observed was a remarkably high andwell-conserved level of CD123 (IL-3R alpha chain) expression amongstprimary AML specimens. FIG. 1 shows representative examples of CD123labeling in normal and leukemic tissue. In FIG. 1A total normal marrow,as well as more primitive subsets, are shown with respect to CD123expression. Total marrow generally has about 7% positive cells forCD123, but only about 1% of the population expresses the antigen at highlevels (see inset FIG. 1A). The CD34+ population of normal marrow alsohas readily evident CD123 expression (12% in FIG. 1A, right histogram),as would be expected for a population known to contain hematopoieticprogenitors. The labeling profile shown is in good agreement withprevious studies by Sato et. al. that have also examined IL-3Rα levelson human CD34+ cells (Sato N, et al., Expression and factor-dependentmodulation of the interleukin-3 receptor subunits on human hematopoieticcells. Blood 1993, 82: 752-61.). However, the more primitive CD34+/CD38−compartment shows no significant expression of CD123 (<1%). In contrast,primary AML cells (FIG. 1B) displayed high levels of CD123. In both theoverall CD34+ population, as well as the more primitive CD34+/CD38−compartment, greater than 99% of the cells were positive for CD123. FIG.2 shows five additional examples of CD123 labeling on CD34+/CD38− AMLcells, further demonstrating the strong expression of this antigen onleukemic populations. Table 1 summarizes the experiments performed todate on the AML cell type, and shows CD123 levels for primitive cells ofAML subtypes M1, M2, and M4. Of the 18 primary AML specimens examined,CD123 was strongly expressed on the primitive leukemia cells in all buttwo instances. The two samples which had lower CD123 levels (samplesAML-11 and AML-14, Table I) both displayed a uniform shift in CD123expression, but had an overall labeling intensity that was dimmer thanmost samples assayed. In many cases (9 of 18), CD123 negative cells werevirtually undetectable (0% or less than 1%). Conversely, expression ofCD123 was not detected on 3 of 5 normal samples of CD34+/CD38− cells andwas barely detectable in two additional specimens (<1%). These flowcytometric analyses were confirmed using two different anti-CD123monoclonal antibodies to insure that the results were not an artifactcaused by the use of a particular antibody.

The high level of CD123 expression found on all AML subtypes examinedimplies that IL-3Rα might play a central role in creating or maintainingthe leukemic state. To form the high affinity receptor for IL-3, boththe α and β chains (CD123 and CD131 respectively) are necessary. Thus,expression of CD131 was also examined by flow cytometry on the AMLspecimens. Interestingly, while some expression was seen in bulk AMLpopulations, in 15 of 15 specimens CD131 was never detected in the CD34+compartment (data not shown).

Further data demonstrating CD123 expression in primary ALL and primaryCML cells, as well as non-Hodgkin's lymphoma, are discussed in Examples8 and 10.

Example 6 In Vivo Engraftment Properties of Human CD123+ Leukemia Cellsin NOD/SCID Mice

Given the strong CD123 expression observed on the vast majority of cellsthat phenotypically encompass the LSC population, it appeared likelythat CD123 would be useful as a marker of LSC's. Therefore, to establishthe functional capacity of CD123+ cells, transplantation studies usingthe NOD/SCID mouse model system were performed. Three primary AMLspecimens (AML-2, 5, and 15 from Table 1) were assayed by flowcytometrically sorting CD34+/CD123+ cells and transplanting them intoirradiated NOD/SCID mice. In addition, the remaining cells in thepopulation (CD34−/CD123+/−) were also sorted and transplanted inparallel. The data in FIG. 3 are a representative example of onespecimen that showed strong engraftment of leukemic cells at six weekspost-engraftment. Panel A shows total bone marrow cells labeled withantibodies specific to human CD34 and CD45. The flow cytometric profileclearly indicates that a large population of human cells (CD45+) ispresent in the marrow. In addition, the population is divided betweenCD34+ and CD34− subsets, similar to the proportions of CD34 labelingseen in the original leukemic specimen. Panel B shows the same marrowsample gated only on the CD45+ cells. The data indicate that all of thecells that have proliferated in vivo are CD123 positive. Table 2summarizes the data for the three specimens tested. In all cases, theCD123+ cells were capable of engrafting the NOD/SCID animals. Moreover,in all but one instance, the CD123− populations did not contribute to invivo repopulation. Thus, as defined by the NOD/SCID model, we concludethat CD123 is expressed on the LSC.

Finally, as an independent means of confirming the leukemic origin ofCD123 positive cells, flow cytometry was used to sort CD34+/CD123+ cellsfrom two leukemic specimens. These samples were cultured for four days,synchronized and then harvested for cytogenetic analysis. Examination ofspreads from each specimen showed that 20 out of 20 metaphases waspositive for the leukemia-specific translocation.

Example 7 Biological Role of CD123 Expression in Leukemia Cells

To further corroborate the data obtained by flow cytometry, immunoblotstudies were performed to analyze IL-3R signal transduction components.For these studies, each AML sample was derived from a peripheral bloodspecimen and was sorted to isolate the CD34+ population, thus insuring avirtually pure leukemic sample. First, expression of both the IL-3Rα andβ chains were examined. With respect to CD123, the data shown in FIG. 4(top panel) clearly demonstrate expression in all leukemic samplesassayed. The CD34+ cells derived from normal marrow (lane 2, CD34+) alsoshow a weak signal. This is consistent with the data in FIG. 1A, whichshow that normal CD34+ cells often contain a small subset of CD123+cells. However, CD123 expression was not detected by flow cytometry inthe more primitive CD34+/CD38− subset of normal cells (FIG. 1A and Table1). Due to their low frequency, it was not possible to obtain sufficientCD34+/CD38− cells of either normal or AML origin for direct analysis byimmunoblot. Nonetheless, detection of a clear signal in the overallCD34+ population corroborates the strong signal seen by flow cytometryfor AML cells. Another point to note is that the molecular weight of theCD123 band appears to vary slightly between AML samples. We haveperformed RT-PCR fingerprint analyses of the same specimens and seen noobvious aberrancies (data not shown). Thus, it appears that varyingdegrees of post-translational modification are the most likelyexplanation for this observation. Consistent with the results obtainedby flow cytometry, expression of CD131 was not detected (FIG. 4, bottompanel).

To begin exploring a potential functional role for CD123, we examinedthe response of primary AML cells to IL-3. Typically, stimulation ofhematopoietic cells with IL-3 leads to several well-characterizedintracellular signal transduction events (Hara T, et al., Function andsignal transduction mediated by the interleukin 3 receptor system inhematopoiesis. Stem Cells 1996, 14: 605-18). Prevalent among theseevents are phosphorylation of Mek-1, Akt, and Stat-5 (Songyang Z, etal., Interleukin 3-dependent survival by the Akt protein kinase. ProcNatl Acad Sci USA 1997, 94: 11345-50; Yagisawa M, et al., Signaltransduction pathways in normal human monocytes stimulated by cytokinesand mediators: comparative study with normal human neutrophils ortransformed cells and the putative roles in functionality and cellbiology. Exp Hematol 1999, 27: 1063-76; Sutor S L, et al., Aphosphatidylinositol 3-kinase-dependent pathway that differentiallyregulates c-Raf and A-Raf. J Biol Chem 1999, 274: 7002-10; de Groot R P,et al., Regulation of proliferation, differentiation and survival by theIL-3/IL-5/GM-CSF receptor family. Cell Signal 1998, 10: 619-28).Consequently, immunoblot studies were performed on these proteins toassess the degree of phosphorylation, both in the presence or theabsence of IL-3 stimulation. The data, shown in FIG. 5, show nodetectable phosphorylation of Akt and Stat5 in the absence of IL-3, andonly a moderate level of phosphorylation for Mek-1. Furthermore, inresponse to IL-3 stimulation, no appreciable increase in phosphorylationis seen for any of the proteins assayed. These results suggest thatCD123 present on the surface of primary AML cells does not contributesignificantly to signal transduction via conventional IL-3 mediatedpathways.

Example 8 CD123 Expression in Primary ALL and Primary CML Cells

Similar experimental protocol as described in Examples 1 through 4 werefollowed, and the expression of CD123 was assayed in primary ALL andprimary CML cells by flow cytometry. The results were consistent withthe results obtained with expression of CD123 in primary AML in Example5. FIGS. 6A, 6B, and 6C show reproducibly that the primary ALL cellsexpress CD123. Moreover, FIGS. 7A, 7B, and 7C show reproducibly that theprimary CML cells also express CD123.

Example 9 CD123 Targeted Complement-Kill Assay

By using a complement-kill assay, “Current protocols in Immunology”Edited by John Coligan, Ada Kruisbeek, David Margulies, Ethan Shevach,and Warren Strober, John Wiley and Sons publishing, 1992, which isincorporated by reference herein in its entirety, this experimentdemonstrates that CD123+ cells are preferentially targeted. In thisexperiment, we compared a typical AML specimen (i.e. CD123+) to a normalbone marrow sample. For each specimen there is an untreated control, asample treated with complement alone, and a sample treated withanti-CD123+ complement. As shown in Table 3, there is a substantialcomplement-killing effect on the AML specimen, but no effect on thenormal marrow. This is true for both the overall sample, as well as themore primitive CD34+ cells. Accordingly, this experiment demonstratesthat there is a difference between the effect on normal and leukemiccells with respect to the specificity for CD123.

Example 10 CD123 Expression in Lymphoma Cells

Immunohistochemical analysis of tissue sections showed that “diffuselarge B cell lymphoma” is strongly positive for expression of CD123.This is the most common form of non-Hodgkin's lymphoma. Thus, in aB-cell derived cancer, expression of CD123 is consistent with theobservation that CD123 was also observed on ALL cells (i.e. another typeof B cell cancer). This observation directly identifies B cell lymphomasas a target for therapies and diagnostics using CD123.

All of the cited references are incorporated by reference herein intheir entirety.

Those skilled in the art will recognize, or be able to ascertain usingno more than routine experimentation, many equivalents to the specificembodiments of the invention specifically described herein. Suchequivalents are intended to be encompassed in the scope of the followingclaims.

TABLE 1 Expression of CD123 in Primitive Leukemic and Normal Cells. %CD123+ cells in Specimen FAB CD34+/CD38− population AML AML-1 MDS/AML 100% AML-2 M1  100% AML-3 M1 95.2% AML-4 M4 98.1% AML-5 M2 99.7% AML-6M4 99.5% AML-7 nd  100% AML-8 M4 99.7% AML-9 M4 99.8% AML-10 M2  100%AML-11 MDS/AML 70.2% AML-12 nd 92.5% AML-13 M1 97.5% AML-14 M4 51.1%AML-15 M4 98.1% AML-16 M1 95.3% AML-17 M1 98.9% AML-18 M4  100% NormalMarrow BM-1 na 0 BM-2 na   <1% BM-3 na 0 BM-4 na 0 BM-5 na   <1% FAB =French, American, British classification system MDS/AML =myelodysplastic syndrome progressing to AML na = not applicable

TABLE 2 Engraftment of CD123+ Populations in NOD/SCID Mice. PopulationCells % CD45+ Exp. Specimen Assayed (N) Inj./mouse cells/recip. 1 AML-2CD34+/CD123+ (3) 4.9 × 10e6  42%  18%  67% CD34−/CD123+/− (3) 7.5 × 10e5nd 0.2% 0.1% 2 AML-5 CD34+/CD123+ (4) 2.5 × 10e6  15%   6%   1%  12%CD34−/CD123+/− (3) 2.5 × 10e6 nd nd nd 3 AML-15 CD34+/CD123+ (5) 2.1 ×10e6 2.1% 0.2% 0.8% 1.1% 0.9% CD34−/CD123+/− (3) 2.4 × 10e6 nd nd nd N =number of mice assayed nd = not detectable

TABLE 3 CD123 Specific Complement-Kill Assay Viability Total Cells CD34+Primary AML specimen unstained control 68.53% 73.29% complement only67.76% 77.17% anti-CD123 + 21.31% 33.25% complement Primary normal BMunstained control 80.20% 78.20% complement only 79.42% 80.33%anti-CD123 + 80.46% 81.51% complement

What is claimed is:
 1. A method for reducing the number of CD123-bearingleukemia cells in a human diagnosed with acute myelogenous leukemia(AML), said method comprising administering to the human atherapeutically effective amount of an antibody that binds CD123, suchthat said administration causes a reduction in the number of leukemiacells in the subject.
 2. The method of claim 1, wherein thetherapeutically effective amount is a dosage of about 0.01 mg/kg toabout 500 mg/kg.
 3. The method of claim 1, further comprising monitoringthe amount of AML cells present in the human to determine thetherapeutic effectiveness of the antibody.
 4. The method of claim 1,further comprising monitoring the amount of CD123-bearing leukemia cellspresent in the human to determine the therapeutic effectiveness of theantibody.
 5. The method of claim 1, wherein the therapeuticallyeffective amount of the antibody is administered to the human daily orfor one or several days.
 6. The method of claim 1, wherein the antibodyis a monoclonal antibody, F(ab′)₂, Fab or Fv.
 7. The method of claim 1,further comprising administering to the human an additional therapeuticagent.
 8. The method of claim 7, wherein the additional therapeuticagent is a chemotherapeutic agent.
 9. The method of claim 8, wherein thechemotherapeutic agent is a steroid, cytosine arabinoside, fluorouracil,methotrexate, aminopterin, an anthracycline, mitomycin C, a vincaalkaloid, demecolcine, etoposide, mithramycin, calicheamicin, CC-1065,chlorambucil, or melphalan.
 10. The method of claim 1, wherein theantibody is administered intravenously, intramuscularly, subcutaneously,percutaneously, orally, parenterally, vaginally, ocularly, nasally,transdermally, or intraperitoneally.